U.S. patent number 8,367,370 [Application Number 12/285,020] was granted by the patent office on 2013-02-05 for droplet-based cell culture and cell assays using digital microfluidics.
The grantee listed for this patent is Irena Barbulovic-Nad, Aaron R. Wheeler. Invention is credited to Irena Barbulovic-Nad, Aaron R. Wheeler.
United States Patent |
8,367,370 |
Wheeler , et al. |
February 5, 2013 |
**Please see images for:
( Certificate of Correction ) ** |
Droplet-based cell culture and cell assays using digital
microfluidics
Abstract
We introduce a method for implementing cell-based assays and
long-term cell culture. The method is based on digital
microfluidics (DMF) which is used to actuate nanoliter droplets of
reagents and cells on a planar array of electrodes. DMF method is
sutable for assaying and culturing both cells in suspension and
cells grown on surface (adherent cells). This method is
advantageous for cell culture and assays due to the automated
manipulation of multiple reagents in addition to reduced reagent
use and analysis time. No adverse effects of actuation by DMF were
observed in assays for cell viability, proliferation, and
biochemistry. These results suggest that DMF has great potential as
a simple yet versatile analytical tool for implementing cell-based
assays and cell culture on the microscale.
Inventors: |
Wheeler; Aaron R. (Toronto,
CA), Barbulovic-Nad; Irena (Toronto, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Wheeler; Aaron R.
Barbulovic-Nad; Irena |
Toronto
Toronto |
N/A
N/A |
CA
CA |
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Family
ID: |
40939206 |
Appl.
No.: |
12/285,020 |
Filed: |
September 26, 2008 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090203063 A1 |
Aug 13, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61064002 |
Feb 11, 2008 |
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Current U.S.
Class: |
435/29;
435/395 |
Current CPC
Class: |
C12M
41/00 (20130101); B01L 3/502792 (20130101); C12M
25/08 (20130101); C12Q 1/02 (20130101); C12M
23/16 (20130101); B01F 13/0071 (20130101); C12M
25/01 (20130101); B01F 13/0076 (20130101); G01N
33/54386 (20130101); C12M 33/00 (20130101); B01L
2300/089 (20130101); B01L 2400/0427 (20130101); B01L
2400/0688 (20130101); B01L 2200/027 (20130101); B01L
2300/0867 (20130101); B01L 2200/0605 (20130101); B01L
2300/0819 (20130101) |
Current International
Class: |
C12Q
1/02 (20060101); C12N 5/02 (20060101) |
Field of
Search: |
;435/29,395 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2007120241 |
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Oct 2007 |
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WO |
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WO 2007/120241 |
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Oct 2007 |
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WO |
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2007136386 |
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Nov 2007 |
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WO |
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2008051910 |
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May 2008 |
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WO |
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Other References
Swinbanks, D.1995. Government backs proteome proposal. Nature, vol.
378, No. 6558, p. 653. cited by examiner .
Zergioti et al. 2005. Femtosecond laser microprinting of
biomaterials. Applied Physics Letters, vol. 86, pp. 163902-1 to
163902-3. cited by examiner .
Link et al., "Electric Control of Droplets in Microfluidic
Devices", Angew. Chem. Int. Ed. 2006, 45, pp. 2556-2560. cited by
applicant .
Moon et al., "An integrated digital microfluidic chip for
multiplexed proteomic sample preparation and analysis by MALDI-MS",
Lab Chip. Sep. 2006/ vol. 6(9), 1213-1219. cited by applicant .
Wheeler et al, "Electrowetting-based microfluidics for analysis of
peptides and proteins by matrix=assisted laser
desorption/ionization mass spectrometry" Anal Chem., Aug. 15, 2004,
vol. 76 (16), 4833-4838. cited by applicant .
Chatterjee et al., "Droplet-based microfluidics with nonaqueous
solvents and solutions", Lab Chip, Feb. 2006, vol. 6(2), 199-206.
cited by applicant .
Marc A. Unger. "Monolithic Microfabricated Valves and Pumps by
Multilayer Soft Lithography," SCIENCE (2000) vol. 288. cited by
applicant .
Hongmei Yu. "A plate reader-compatible microchannel array for cell
biology assays," The Royal Society of Chemistry (2007) Lab Chip
vol. 7, pp. 388-391. cited by applicant .
A.S. Verkman, "Drug Discovery In Academia," Am J Physiol Cell
Physiol (2004) vol. 286, pp. 465-474. cited by applicant .
Jamil El-Ali. "Cells on chips," NATURE (2006) Insight Review, vol.
442. cited by applicant .
Eun Zoo Lee, "Removal of bovince serum albumin using solid-phase
extraction with in-situ polymerized stationary phase in a
microfluidic device," ScienceDirect , Journal of Chromatography A.
(2008) vol. 1187 pp. 11-17. cited by applicant .
Shih-Kang Fan. "Cross-scale electric manipulations of cells and
droplets by frequency-modulated dielectrophoresis and
electrowetting" The Royal Society of Chemistry (2008), Lab Chip
vol. 8, pp. 1325-1331. cited by applicant .
Ting-Hsuan Chen. "Selective Wettability Assisted Nanoliter Sample
Generation Via Electrowetting-Based Transportation," Proceedings of
the Fifth International Conference on Nanochannels, Microchannels
and Minichannels (ICNMM) (Jun. 18-20, 2007). cited by applicant
.
Hsih Yin Tan, "A lab-on-a-chip for detection of nerve agent sarin
in blood," The Royal Society of Chemistry (2008), Lab Chip vol. 8,
pp. 885-891. cited by applicant .
Mohamed Abdelgawad. "Low-cost, rapid-prototyping of digital
microfluidics devices," Springer, Microfluid Nanofluid (2008) vol.
4, pp. 349-355. cited by applicant .
Mais J. Jebrail, "Digital Microfluidic Method for Protein
Extraction by Precipitation," Anal. Chem. (2009) vol. 81, No. 1.
cited by applicant .
Eric Lebrasseur. "Two-dimensional electrostatic actuation of
droplets using a single electrode panel and development of
disposable plastic film card," ScienceDirect, Sensors and Actuators
(2007) vol. 136, pp. 358-366. cited by applicant .
Kai-Cheng Chuang. "Direct Handwriting Manipulation of Droplets By
Self-Aligned Mirror-EWOD Across A Dielectric Sheet," MEMS (Jan.
2006) pp. 22-26. cited by applicant.
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Primary Examiner: Naff; David
Assistant Examiner: Srivastava; Kailash C
Attorney, Agent or Firm: Schumacher; Lynn C. Leonard;
Stephen W. Hill & Schumacher
Parent Case Text
CROSS REFERENCE TO RELATED U.S. APPLICATIONS
This patent application relates to, and claims the priority benefit
from, U.S. Provisional Patent Application Ser. No. 61/064,002 filed
on Feb. 11, 2008, in English, entitled DROPLET-BASED CELL ASSAYS,
and which is incorporated herein by reference in its entirety.
Claims
Therefore what is claimed is:
1. A method of performing droplet-based cell culture, comprising
the steps of: a) providing a digital microfluidic device
comprising: an array of actuating electrodes formed on a substrate
surface; and a coating formed on the substrate surface, the coating
providing a working surface; wherein said actuating electrodes are
connectable to an actuating electrode controller for exciting or
de-exciting the actuating electrodes for translating liquid
droplets over the working surface; b) providing a cell-containing
droplet at a location on the working surface, the cell-containing
droplet containing cells and cell culture medium; c) incubating the
digital microfluidic device in a controlled environment to culture
the cells; d) actuating electrodes of the array to dispense a
droplet containing cell culture media to the cell-containing
droplet; and e) incubating the digital microfluidic device in the
controlled environment to further culture the cells.
2. The method according to claim 1, wherein steps d) and e) are
performed one or more additional times.
3. The method according to claim 1, wherein step d) further
comprises actuating electrodes of the array and splitting the cell
containing droplet into at least two smaller cell-containing
droplets.
4. The method according to claim 3, wherein step d) is repeated one
or more times.
5. The method according to claim 4 wherein step d) is performed a
sufficient number of times to generate at least one smaller
cell-containing droplet containing a single cell.
6. The method according to claim 1, wherein the cells include
adherent cells, and wherein the location in step b) includes a cell
culture site adapted for adhesion and proliferation of adherent
cells, such that the adherent cells adhere to the cell culture site
during steps c) to e).
7. The method according to claim 6, wherein step b) includes:
actuating electrodes of the array to transport one or more droplets
containing adherent cells and cell culture media to the location on
the working surface, thereby providing the cell-containing droplet
at the location on the working surface, such that the adherent
cells adhere to the cell culture site.
8. The method according to claim 7, wherein actuating electrodes of
the array to transport one or more droplets containing adherent
cells and cell culture media to the location on the working surface
includes actuating electrodes of the array to dispense a
cell-containing droplet larger than the cell culture site over the
cell culture site, thereby passively dispensing a smaller
cell-containing droplet on the cell culture site.
9. The method according to claim 6, wherein in step d), the droplet
containing cell culture medium is dispensed to the cell-containing
droplet such that the droplet on the cell culture site is replaced
by at least a portion of the droplet containing cell culture media,
such that the adherent cells attached to the cell culture site are
submersed in the cell culture medium.
10. The method according to claim 9, wherein steps d) and are
performed one or more additional times.
11. The method according to claim 9 further comprising performing
one or more cell assays on the adherent cells after culturing the
adherent cells.
12. The method according to claim 11, wherein performing the one or
more cell assays on the adherent cells after culturing the adherent
cells includes detecting signals emitted from the cell containing
droplet using a device capable of detecting a signal from a
cell-containing droplet selected from the group consisting of
optical sensors, optical detectors comprising a light source and a
photodetector, optical detectors that measure any one or
combination of absorbance, fluorescence, epifluorescence, and
chemiluminescence, UV light detectors, radiometric detectors, any
one of scanning, imaging, and confocal microscopy detectors, CCD
cameras, and microplate readers.
13. The method according to claim 11, wherein performing one or
more assays includes actuating electrodes of the array to dispense
one or more assay reagents to the adherent cells.
14. The method according to claim 9, wherein in step d), the
droplet containing cell culture media has a base area larger than
the cell culture site, and wherein after actuating electrodes of
the array to dispense the droplet containing cell culture media to
the cell-containing droplet, the method further comprises actuating
electrodes of the array to remove a droplet from a region including
the cell culture site, such that a smaller droplet containing cell
culture media remains behind on the cell culture site.
15. The method according to claim 14, wherein steps d) and e) are
performed one or more additional times.
16. The method according to claim 14 wherein the cell culture site
is hydrophillic.
17. The method according to claim 9 further comprising: translating
one or more droplets containing a washing solution over one or more
cell culture sites; dispensing one or more droplets containing a
cell dissociation agent to the cell culture sites; and incubating
the digital microfluidic device for a time duration sufficient to
detach the adherent cells from the cell culture site, thereby
forming a droplet containing resuspended cells.
18. The method according to claim 17 further comprising dispensing
one or more additional droplets to said droplet containing
resuspended cells, wherein said one or more additional droplets
contain cell culture media.
19. The method according to claim 6, wherein the cell culture site
is prepared by depositing a bio-substrate on the working
surface.
20. The method according to claim 19, wherein said bio-substrate is
deposited using any one of microprinting and microstamping.
21. The method according to claim 19, wherein said bio-substrate is
produced from cell specific constituents.
22. The method according to claim 21, wherein said cell specific
constituents are extracellular matrix proteins.
23. The method according to claim 22, wherein said extracellular
matrix proteins include any one of fibronectin, laminin, collagen,
elastin and any combination thereof.
24. The method according to claim 21, wherein said cell specific
constituents are synthetic molecules comprised of one of
poly-L-lysine, poly-D-lysine and any combination thereof.
25. The method according to claim 6, wherein the coating includes
any one of a hydrophobic layer and a dielectric layer, and wherein
the cell culture site is prepared using any one or combination of
plasma treatment, hydrophobic layer etching, dielectric layer
etching, electrode etching and stamping.
26. The method according to claim 6 wherein the cell culture site
is hydrophillic.
27. The method according to claim 1, wherein step b) is performed
by actuating electrodes of the array to transport one or more
cell-containing droplet and droplet containing cell culture medium
to the location on the working surface.
28. The method according to claim 1, wherein step b) is performed
by transporting one or more droplets from one or more sources
external to said digital microfluidic device.
29. The method according to claim 28, wherein the one or more
sources external to said digital microfluidic device are selected
from the group consisting of pipettes, robotic dispensers,
microprinters and microstamps.
30. The method according to claim 1, wherein step b) is performed
by dispensing one or more droplets containing cells and cell
culture media from one or more sources integrated as part of said
digital microfluidic device, said one or more sources being in flow
communication with said working surface.
31. The method according to claim 30, wherein the one or more
sources integrated as part of said digital microfluidic device are
liquid reservoirs.
32. The method according to claim 31, wherein the liquid reservoirs
are formed on said working surface above some of said actuating
electrodes which are modified to act as said liquid reservoirs.
33. The method according to claim 1, wherein step b) is performed
by dispensing one or more droplets containing cells and cell
culture media from one or more sources integrated as part of a
cartridge assembled with said digital microfluidic device, said one
or more sources being in flow communication with said working
surface.
34. The method according to claim 1, wherein the cell-containing
droplet is a combination of cells, a suspension medium, and a
non-ionic surfactant.
35. The method according to claim 34, wherein said suspension
medium is selected to facilitate cell-containing droplet actuation
by preventing non-specific adsorption of cells and proteins to
device surfaces.
36. The method according to claim 34, wherein the cell-containing
droplet is a combination of cells and a suspension medium selected
from the group consisting of block copolymers formed from
poly(propylene oxide) and poly(ethylene oxide), poloxamer 188,
poloxamer 407, hydrophilic polymers, sodium bicarbonate, phosphate
buffered saline (PBS), HEPES, and cell culture medium selected from
the group consisting of balanced salt solutions, nutrient mixtures,
basal media, complex media, serum free media, insect cell media,
virus production media, serum, fetal bovine serum, serum
replacements, antibiotics, antimycotics, and any combination
thereof.
37. The method according to claim 34, wherein the cell-containing
droplet is a combination of cells, phosphate buffered saline, and
poloxamer 188.
38. The method according to claim 1, further comprising actuating
electrodes of the array to dispense at least one additional droplet
to the cell-containing droplet, wherein the additional droplet
contains a substance selected from the group consisting of
chemicals, biochemicals, drugs, drug lead compounds, toxins,
surfactants, transfection reagents, supplements, anti-clumping
agents, streptavidin, biotin, antibody production enhancers,
antibodies, antibody ligands, nucleic acids, nucleic acid binding
molecules, enzymes, proteins, viruses, cell process agonists or
antagonists, labeling agents, fluorescent dyes, fluorogenic dyes,
viability dyes, calcein AM, quantum dots, nano particles,
polysorbate 20, ethidium homodimer-1, block copolymers formed from
poly(propylene oxide) and poly(ethylene oxide), poloxamer 188,
poloxamer 407, hydrophilic polymers, sodium bicarbonate, phosphate
buffered saline (PBS), HEPES, and cell culture medium selected from
the group consisting of balanced salt solutions, nutrient mixtures,
basal media, complex media, serum free media, insect cell media,
virus production media, serum, fetal bovine serum, serum
replacements, antibiotics, antimycotics, and any combination
thereof.
39. The method according to claim 1, wherein the cells include
primary/isolated or transformed/cultured cells selected from the
group consisting of prokaryotic cells, eukaryotic cells, animal
cells, blood cells, human leukemia cells, lymphocytes, beta cells,
oocytes, eggs, primary cells, primary bone marrow cells, stem
cells, neuronal cells, endothelial cells, epithelial cells,
fibroblasts, insect cells, plant cells, bacterial cells, and
archaebacterial cells.
40. The method according to claim 1, wherein the cell containing
droplet has a cell density less than about 1.times.10.sup.3
cells/mL.
41. The method according to claim 1 conducted in a substantially
sterile chamber.
42. The method according to claim 41 further comprising translating
at least some cells mixed with the cell culture media to at least
one new cell culture site to seed a new generation of cells.
43. The method according to claim 1, wherein the environment is
controlled by regulating conditions including humidity, temperature
and atmosphere.
44. The method according to claim 1 performed in a multiplexed
mode, wherein: said step b) of providing a cell-containing droplet
at a location on the working surface includes providing a plurality
of cell-containing droplets at a plurality of locations on the
working surface, each cell-containing droplet containing cells and
cell culture media; and wherein said step d) of actuating
electrodes of the array to dispense a droplet containing cell
culture media to the cell-containing droplet includes actuating
electrodes of the array to dispense a plurality of droplets,
containing cell culture media to the cell-containing droplets such
that at least one droplet containing cell culture media is
dispensed to each cell-containing droplet.
45. The method according to claim 44, wherein the plurality of
droplets containing cell culture medium is equal in number to the
plurality of cell-containing droplets.
46. The method according to claim 44, wherein each of said
plurality of cell-containing droplets includes cell suspensions
identical to cell suspensions in the rest of said droplets, and
wherein step b) is performed by providing the plurality of
cell-containing droplets from one source.
47. The method according to claim 44, wherein each of said
plurality of cell-containing droplets includes cell suspensions
different to the cell suspensions in the rest of the
cell-containing droplets, and wherein step b) is performed by
dispensing the plurality of cell-containing droplets from a
corresponding plurality of sources, each of said plurality of
sources having a cell suspension different from the rest of the
cell suspensions.
48. The method according to claim 44, wherein the cells include
adherent cells, and wherein prior to step b), one or more of said
plurality of locations are modified to produce a plurality of cell
culture sites, and wherein adherent cells adhere to said plurality
of cell culture sites.
49. The method according to claim 44, wherein the cells include
adherent cells, and wherein said each location in step b) includes
a cell culture site adapted for adhesion and proliferation of
adherent cells, such that the adherent cells adhere to the cell
culture site during steps c) to e).
50. The method according to claim 44, wherein the plurality of
droplets containing cell culture medium are dispensed to the
cell-containing droplets such that each droplet containing cell
culture medium mixes with, or displaces and replaces, the
respective cell-containing droplet.
51. The method according to claim 1,wherein steps b) to e) are
conducted according to a cell culture protocol under control of a
computer controller interfaced to said digital microfluidic
device.
52. The method according to claim 1 further comprising performing
one or more cell assays on the cell containing droplet after
culturing the cells.
53. The method according to claim 52, wherein performing the one or
more cell assays on the cell-containing droplet after culturing the
cells includes detecting signals emitted from the cell containing
droplet using a device capable of detecting a signal from a
cell-containing droplet selected from the group consisting of
optical sensors, optical detectors comprising a light source and a
photodetector, optical detectors that measure any one or
combination of absorbance, fluorescence, epifluorescence, and
chemiluminescence, UV light detectors, radiometric detectors, any
one of scanning, imaging, and confocal microscopy detectors, CCD
cameras, and microplate readers.
54. The method according to claim 52 wherein at least a portion of
the cells in the cell-containing droplet are alive when the one or
more cell assays are performed.
55. The method according to claim 52, wherein performing one or
more assays includes actuating electrodes of the array to dispense
one or more assay reagents to the cell-containing droplet.
Description
FIELD OF THE INVENTION
The present invention relates to droplet-based cell assays and/or
cell culture using digital microfluidics, and more particularly,
the present invention relates to devices and methods used with
those devices for performing cell assays and/or cell culture.
BACKGROUND OF THE INVENTION
The cell is the irreducible element of life and is often studied as
a living model of complex biological systems. Cell-based assays are
conventionally performed in well plates that enable simultaneous
analysis of multiple cell types or stimuli. For such multiplexed
analyses, cells in well plates are often evaluated using microplate
readers, which can be integrated with fluid handling and other
miscellaneous equipment in a robotic analysis platform. A major
drawback of such systems is the expense of the instrumentation and
the experimental consumables (e.g., plates, pipette tips, reagents,
and cells). The latter is a particular disadvantage for cell-based
assays as they are generally more complex and require larger
amounts of reagents than cell-free assays..sup.1
Recently, microfluidics has been touted as a solution for the
challenges inherent in conducting multiplexed cell-based
assays..sup.2 The conventional format for microfluidics, which is
characterized by devices containing networks of micron-dimension
channels, allows integration of multiple processes on a single
platform while reducing reagent consumption and analysis time.
There are numerous advantages of using microfluidic based systems
for cell assays, some of which are self-similarity in dimensions of
cells and microchannels (10-100 .mu.m widths and depths), laminar
flow dominance and formation of highly resolved chemical gradients,
subcellular delivery of stimuli, reduced dilution of analytes, and
favorable scaling of electrical and magnetic fields. For the last
ten years, researchers have used microchannels to manipulate and
sort cells, to analyze cell lysates, to assay intact-cell
biochemistry, and to evaluate cell mechanical and electrical
responses. In most of these studies, cells were exposed to one
stimulus or to a limited number of stimuli. There have been just a
few attempts to conduct multiplexed assays as it is difficult to
control many reagents simultaneously in a complex network of
connected channels, even when using microvalve architectures
developed for microfluidic devices..sup.3 Finally, we note that
there have been only a few microfluidic devices integrated to
multiplexed detection instruments such as microplate readers;.sup.4
we believe this will be a necessary step for the technology to
become competitive with robotic screening systems.
A potential solution to the limitations of the channel-microfluidic
format is the use of "digital" or droplet-based microfluidics. In
digital microfluidics (DMF), discrete droplets containing reagents
are manipulated by sequentially applying potentials to adjacent
electrodes in an array..sup.5-14 Droplets can be manipulated
independently or in parallel on a reconfigurable path defined by
the electrode actuation sequence, which allows for precise spatial
and temporal control over reagents. As with all microscale
techniques, cross-contamination is a concern for DMF, but this
phenomenon can be avoided by dedicating separate paths for each
reagent. DMF has been used to actuate a wide range of volumes (nL
to .mu.L) and, unlike channel devices, there is no sample wasted in
creating small plugs for analysis. In addition, each droplet is
isolated from its surroundings rather than being embedded in a
stream of fluid--a simple method of forming a microreactor in which
there is no possibility that products will diffuse away. The
preservation of products in a droplet is of great importance in
cell assays targeting molecules secreted from cells into
extracellular space. In addition, droplets provide mostly static
fluid conditions without unwanted shear stress that is inevitable
in continuous flow microfluidics. A further advantage of DMF is its
capacity to generate nanoliter samples by translating droplets
through selective wettability areas on an electrowetting-based
platform..sup.15
There is currently much enthusiasm for using DMF to implement
multiplexed assays; however, it has only been applied to a few
non-cell assays. To the inventors' knowledge, there are no reports
of the use of DMF to analyze cells. There are a few studies
demonstrating only dispensing and manipulation of droplets
containing cells, cell sorting, and cell concentration on a DMF
platform. WO 2007/120241 A2 entitled "Droplet-Based
Biochemistry".sup.16 discloses dispensing and dividing droplets
containing cells, generating droplets with single cells, detecting
a type of cell, and sorting cells. US20070148763 A1 entitled
"Quantitative cell dispensing apparatus using liquid drop
manipulation".sup.17 describes cell droplet handling, to achieve a
predetermined number of cells. In a journal paper by Fan et
al,.sup.18 dielectrophoresis was used to concentrate neuroblastoma
cells within droplets on a DMF platform.
It would be very advantageous to provide droplet-based cell culture
and/or assays using digital microfluidics in order to enable
automated cell micro culture and high-throughput screening ability
for cell analysis. DMF would also address some problems associated
with standard culture and assaying in well-plates or in
continuous-flow microfluidic devices.
SUMMARY OF INVENTION
The present invention provides embodiments of devices and methods
for droplet-based cell culture and assays using digital
microfluidic devices designed to manipulate, operate, and analyze
cell-containing droplets. Cells in a suspension and cell-assay
and/or cell-culture reagents are deposited in the device by either
dispensing them from device reservoirs or dispensing them into the
device using external means (e.g., pipette, robotic dispenser,
etc.). In order to perform an assay with cells in suspension,
cell-containing droplets and reagent-containing droplets are moved
between adjacent electrodes by applying voltages to electrodes.
General assay protocol comprises dispensing and translating
droplets, merging and mixing droplets with cells and reagents at
least once, possible splitting of droplets, incubating cells with
reagents in merged/mixed (and split) droplets at least once, and
detecting signal from cells in merged/mixed (and split) droplets in
the device after final incubation. Using the same DMF techniques,
suspended cells are also long-term cultured and split at regular
time intervals.
Additionally, DMF devices are designed to culture and assay
adherent cells. After being introduced in a device in suspension,
adherent cells are seeded on cell culture sites (patterned DMF
device surface for cell attachment), where they can be long-term
cultured in droplets, subcultured using standard subculture
protocols, and assayed. Media exchange and regent delivery on cell
culture sites (CSSs) is performed using standard DMF operations:
translating, merging, mixing and splitting droplets. In addition, a
new technique, passive dispensing, is developed for more efficient
delivery of reagents/media from big source droplet translating over
CCSs. By means of DMF and passive dispensing, a first
multigenerational cell culture in a microscale is realized.
Culture and assay reagents comprise chemical, biochemical and
biological reagents. Droplets contain additives including pluronics
and various hydrophilic polymers to facilitate cell-containing
droplet actuation by preventing non-specific adsorption of cells
and proteins to a device surface.
In a multiplexed assay, multiple cell-containing droplets (which
may include one kind or multiple kinds of cells) are manipulated
and assayed simultaneously or in a certain sequence with one or
multiple reagents.
Thus, in an embodiment of the present there is provided method of
performing droplet-based cell culture, comprising the steps of:
a) providing a digital microfluidic device comprising: an array of
actuating electrodes formed on a substrate surface; and a coating
formed on the substrate surface, the coating providing a working
surface; wherein said actuating electrodes are connectable to an
actuating electrode controller for exciting or de-exciting the
actuating electrodes for translating liquid droplets over the
working surface;
b) providing a cell-containing droplet at a location on the working
surface, the cell-containing droplet containing cells and cell
culture media;
c) incubating the digital microfluidic device in a controlled
environment to culture the cells;
d) actuating electrodes of the array to dispense a droplet
containing cell culture media to the cell-containing droplet;
and
e) incubating the digital microfluidic device in the controlled
environment to further culture the cells.
In another aspect of the present invention there is provided a
digital microfluidic device for conducting one or both of cell
assays and cell culture, comprising: a first substrate having a
first substrate surface; an array of actuating electrodes formed on
the first substrate surface; at least one dielectric layer formed
on the first substrate surface covering each actuating electrode
such that the actuating electrodes are electrically insulated from
one another; and at least one reference electrode, wherein each
actuating electrode is proximal to at least one of the reference
electrodes; an electrode controller capable of selectively exciting
or de-exciting actuating electrodes for translating liquid droplets
across a surface of the dielectric layer; one or more first
reservoirs in flow communication with the surface of said
dielectric layer for holding at least one suspension of cells and
one or more reagent reservoirs in flow communication with the
surface of said dielectric layer for holding one or more cell assay
reagents, cell culture reagents; and dispensing means for
dispensing droplets of said at least one suspension of cells and
droplets of said at least one cell assay reagents, cell culture
reagents onto said surface of said dielectric layer; and a computer
controller interfaced to said dispensing means and said electrode
controller and being programmed to dispense droplets of the
suspension of cells and droplets of said one or more cell assay
reagents, cell culture reagents onto said surface of said
dielectric layer and translating them over said array of actuating
electrodes for mixing and optionally splitting said droplets in
selected positions on said array of actuating electrodes to form
one or more secondary droplets in a selected order defined by a
selected cell assay protocol or cell culture protocol for which
said computer controller is programmed.
A further understanding of the functional and advantageous aspects
of the invention can be realized by reference to the following
detailed description and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention will now be described, by
way of example only, with reference to the drawings, in which:
FIG. 1 is a top view of a complete digital-microfluidic device
showing three droplet sources: cells, reagent, and dye;
FIG. 2(a) shows a cross-sectional view of the device of FIG. 1;
FIG. 2(b) shows a cross sectional view of an alternative embodiment
of the device of FIG. 1 which uses a one-plate design;
FIGS. 3(a) to (c) show three frames from a movie wherein a droplet
with cells is dispensed from a reservoir;
FIG. 4 is a plot of numerically simulated potential drops across a
droplet and a dielectric layer;
FIG. 5 is a graph of viability and proliferation tests for cells
actuated by digital microfluidics showing no significant
differences between the actuated and non-actuated cells;
FIGS. 6(a) and (b) are graphs of vitality tests wherein cells in
droplets were actuated, lysed, and analyzed by Matrix Assisted
Laser Desorption Ionization Mass Spectrometry (MALDI-MS) showing no
major qualitative differences between the (a) actuated and (b)
non-actuated cells;
FIGS. 7(a) to (f) show sequential images from a movie depicting a
digital microfluidic cell-based assay;
FIGS. 8(a) and (b) show fluorescent images of droplets with cells
treated with (a) 0% and (b) 0.5% Tween 20 and stained with
viability dyes. (calcein AM and ethidium homodimer-1); in the
droplet (a), almost all cells were live (dead cells in (a) are
marked with small circles), and in the droplet (b), all cells were
dead;
FIGS. 9(a) and (b) show two dose-response curves for Jurkat T-cells
exposed to Tween 20 (0.002% to 0.5% (v/v)) using (a) a digital
microfluidics assay and (b) a well-plate assay;
FIG. 10 shows a top view of an embodiment of a DMF device for
multiplexed cell assays which comprises reservoirs for four
different cell suspensions and nine different assay reagents, and a
waste reservoir;
FIGS. 11(a) to (d) are diagrammatic representations of seeding
adherent cells in a DMF device where (a) shows actively dispensed
droplet of cell suspension translating to a cell culture site
(CCS), (b) shows passively dispensing a droplet of cell suspension
onto the CCS from a source droplet, (c) shows cells in suspension
seeded on the CCS, and (d) shows cell monolayer formed on the ECM
substrate on the CCS;
FIG. 12 is a diagrammatic representation showing passive dispensing
of a droplet where a source droplet provides a smaller liquid
droplet located on the CCS;
FIG. 13 shows several examples of the hydrophilic area positions
relative to actuating electrodes and to the source droplet
path;
FIG. 14 shows a diagrammatic representation showing a passive
washing/exchange process whereby a droplet on a CCS is replaced by
a new droplet;
FIG. 15 shows a graph of fluorescein fluorescence signal intensity
versus washing cycle to show washing efficiency;
FIG. 16 shows a digital image of .about.130 mouse fibroblast cells
(NIH-3T3) cultured in a DMF device for 72 h; media was replenished
using passive dispensing/exchange technique every 24 h; after 72 h
cells were stained with calcein AM for viability;
FIGS. 17(a) to (f) are diagrammatic representations of subculturing
adherent cells in a DMF device in which (a) shows monolayer of
adherent cells cultured on a CCS, (b) washing cells via passive
exchange, (c) delivering a dissociation agent to cells via passive
exchange, (d) detachment of cells after incubation with a
dissociation agent, (e) blocking of a dissociation agent and
resuspending cells via passive exchange, and (f) seeding of cells
resuspended in fresh media on a new CCS;
FIGS. 18(a) to (d) show diagrammatic representations of assaying
adherent cells in a DMF device where, (a) shows a monolayer of
adherent cells cultured on a CCS in cell culture media, (b) washing
cells and delivering assay reagents to cells via passive exchange,
(c) incubating cells with assay reagents, and (d) detecting and
analyzing cell response to assay stimuli; and
FIG. 19 shows a DMF device for multiplexed cell assays with
adherent cells using passive dispensing and passive reagent
exchange.
DETAILED DESCRIPTION OF THE INVENTION
Without limitation, the majority of the systems described herein
are directed to methods and devices for droplet-based cell assays
using digital microfluidics. As required, embodiments of the
present invention are disclosed herein. However, the disclosed
embodiments are merely exemplary, and it should be understood that
the invention may be embodied in many various and alternative
forms.
The figures are not to scale and some features may be exaggerated
or minimized to show details of particular elements while related
elements may have been eliminated to prevent obscuring novel
aspects. Therefore, specific structural and functional details
disclosed herein are not to be interpreted as limiting but merely
as a basis for the claims and as a representative basis for
teaching one skilled in the art to variously employ the present
invention. For purposes of teaching and not limitation, the
illustrated embodiments are directed to droplet-based cell assays
and culture using digital microfluidics (DMF).
As used herein, the term "about" and the symbol ".about.", when
used in conjunction with ranges of dimensions, temperatures or
other physical and/or chemical properties and/or characteristics is
meant to cover slight variations that may exist in the upper and
lower limits of the ranges of dimensions as to not exclude
embodiments whereon average most of the dimensions are satisfied
but where statistically dimensions may exist outside this region.
For example, in embodiments of the present invention dimensions of
a digital microfluidic device are given but it will be understood
that these are not meant to be limiting.
FIG. 1 shows a top view of a microfluidic device shown generally at
10 which may be used for droplet-based cell culture and cell assays
using digital digital microfluidics in accordance with the present
invention. Reservoir electrodes 32, 34, and 36 store droplets 42,
44, 46 containing cells, reagent, and dye, respectively, and are
capable of dispensing the liquids onto the center region 38 of the
device. Small volumes of liquids are dispensed as droplets and
translated by applying voltages to actuating electrodes 14. There
is also another reservoir electrode 30 shown in the device in FIG.
1 which may be used as a reservoir as well.
FIG. 2(a) is a cross-sectional view of a portion of the
microfluidic device 10 of FIG. 1 showing two adjacent electrodes 14
of the electrode array. Electrodes 14 (10 nm Cr+, 100 nm Au) rest
on a substrate layer 12 and are separated from each other by a
dielectric material 16 (for example 2 .mu.m Parylene-C). The device
can have more than one dielectric layer 16. Located on top of
dielectric material 16 is a hydrophobic layer 18 (for example
Teflon AF, 50 nm). The array of actuating electrodes and exposed
areas of substrate surface are thus covered by a working surface.
Spaced above electrodes 14/dielectric layer 16 is a continuous
reference electrode 22 coated on a substrate layer 24, and a
hydrophobic layer 20 (for example Teflon AF, 50 nm) is coated on
reference electrode 22. Alternatively, another dielectric layer can
be deposited between layers 20, 22. Liquid droplets 42 rest
in-between two hydrophobic layers 18 and 20. Electrodes 14, voltage
source 26, and the continuous reference electrode 22 together form
an electric field, digitally manipulated by controller 28. For
droplet manipulation, reference electrodes 22 are biased to a
potential different from the actuating potential. Commonly used
reference potential is ground.
In a preferred embodiment of the present invention, the upper
hydrophobic layer 20, reference electrode 22, and substrate layer
24 are substantially transparent to allow optical analysis of the
assays. Furthermore, layers 20, 22, and 24 are not necessary to
translate droplets.
While the present invention discusses the two-plate design of FIG.
2(a), a one-plate design is also possible, as shown in FIG. 2(b).
In FIG. 2(b), layers 20, 22, and 24 are removed. Rather than have a
dedicated reference electrode layer 22, the reference electrode is
patterned adjacent to electrodes 14, forming a continuous grid 52
separated from electrodes 14 by dielectric material 16. The
continuous grid 52 extends in both directions defining the plane in
which electrodes 14 are located.
Reference electrodes can also be coplanar with the top surface of
the dielectric layer. In a device with multiple dielectric layers,
reference electrodes can be coplanar with the top surface of any
dielectric layer, while being insulated from actuating electrodes
14. The design of reference electrodes is not limited to a grid,
e.g. they can be in a form of a wire or an array similarly to
electrodes 14.
FIG. 3 shows three frames from a movie wherein a 150 nL droplet 42
containing .about.260 cells is dispensed from a reservoir of a
microfluidic device with identical dimensions but fewer electrodes
than the microfluidic device 10 shown in FIG. 1, wherein cells were
labeled with a viability dye, calcein AM, which fluoresces
green.
FIGS. 7(a) to (f) show sequential images from a movie depicting a
digital microfluidic cell-based assay, wherein a 150 nL droplet 42
containing .about.525 cells was dispensed (a, 402), translated (b,
404), and merged (c, 406) with a 150 nL droplet 44 of Tween 20
dispensed (b, 402) from a second reservoir. The merged droplet was
actively mixed (408) on four neighboring electrodes (d); after 20
min incubation in a humidified environment, the combined droplet
was merged (e, 406) and mixed (e, 408) with a 150 nL droplet 46
containing viability dyes. The final droplet was incubated (f, 410)
for 20 minutes in a humidified environment.
A sample result of the microfluidic cell-based assay of FIG. 7(f)
is shown in FIGS. 8(a) and (b), wherein fluorescent images of
droplets treated with (a) 0% and (b) 0.5% Tween 20. Calcein AM
(green) was used to stain live cells, and ethidium homodimer-1
(red) for dead cells. In the former droplet (a), almost all cells
were live (dead cells in (a) are marked with small circles), and in
the latter (b), all cells were dead.
While digital microfluidics has been used previously to manipulate
and evaluate a wide range of liquids and reagents, we report herein
the first application of digital microfluidics to transport,
analyze and culture biological cells. Using the parameters reported
in the experimental section (elaborated below), cell suspensions
representing a wide range of concentrations (including very dense
solutions of 1.times.10.sup.8 cells/mL) were found to be feasible
to be actuated by DMF, with no differences observed in velocity or
reliability relative to liquids not containing cells.
For example, FIGS. 3(a) to (c) depict a routine operation in our
experiments: dispensing of a 150 nL droplet containing .about.260
Jurkat T-cells. However, in initial work (with un-optimized
parameters), droplets containing cells were difficult to
manipulate, as cells tended to stick to the surface of the devices,
causing contact line pinning. This problem was overcome by the use
of the non-ionic surfactant, pluronic F68, which when used as a
solution additive, facilitated actuation of suspensions of cells in
all liquids tested (including PBS and complete media containing 10%
fetal bovine serum).
Pluronics are block copolymers formed from poly(propylene oxide)
(PPO) and poly(ethylene oxide) (PEO), and are commonly used as
surface coatings for preventing non-specific protein adsorption. In
our work, we used pluronics in solution, rather than as a surface
coating; we hypothesize that in this configuration, the polymer
coats cells and proteins in a manner such that their functionality
is retained, but adsorption to hydrophobic surfaces is minimized.
We note that pluronic F68 has been used extensively in cell-based
assays with no evidence for detrimental effects on cell
vitality,.sup.19,20 and it is even used as a constituent in
commercial cell growth media..sup.21 Our experiments support this
trend--Jurkat T-cells incubated in medium containing 0.2% (wt/vol)
F68 for 4 days (humidified incubator, 5% CO2, 37.degree. C.) had
identical growth rates and morphology as cells grown in media
without pluronics. In on-going work, the optimal conditions
(concentration and type of pluronic, etc.) for reducing unwanted
adsorption in DMF are being evaluated; we used F68 for all of the
results reported here.
A second challenge for using DMF for actuation of cells is droplet
evaporation, which raises the concentration of salts and other
buffer constituents, making the solution hypertonic. In the work
described here, we controlled evaporation by positioning devices in
a humidified atmosphere when not actively manipulating droplets by
DMF. For the duration of the assay experiments (up to a few hours),
such measures prevented significant evaporation, and no negative
effects on cell viability were observed. For culturing cells,
devices were placed in cell culture incubators at 37.degree. C. and
5% CO.sub.2. The DMF devices may be contained in a sterile,
humidified chamber for the full duration of the assay or cell
culture process (including actuation, incubation, and analysis) or
culture which facilitates long-term cell culture and
examination.
Effects of DMF Manipulation on Cell Vitality.
Digital microfluidic devices use electrical fields to actuate
droplets, which led us to investigate the effects of droplet
actuation on cell vitality. As described above, droplets are
translated by an energized actuating electrode 14 on a bottom plate
and a reference electrode 22 on a top plate (FIG. 2(a)). It should
be noted that the reference electrode may also be placed on the
bottom plate, as in reference electrode 52 (FIG. 2(b)). Because of
the high conductivity of a droplet 42 of phosphate buffered saline
(PBS) relative to the insulating dielectric layer 16 formed from
Parylene-C, the inventors believe that cells would experience
negligible electrical field upon application of driving potentials.
This hypothesis was supported by a numerical simulation using the
COMSOL Multiphysics 3.3a analysis package. In a simulation, shown
in FIG. 4, in which 100 V was applied between top and bottom
electrodes, the potential drop in the droplet was found to be only
3.73.times.10.sup.-8 V, or 0.00000004% of the applied potential.
Thus, it is contemplated that one would expect to observe modest
effects (if any) on the vitality of suspensions of cells, upon
application of electrical field. These effects were evaluated by
three tests, measuring cell viability, proliferation, and
biochemistry.
As shown in FIG. 5, the viability of actuated and non-actuated
cells was compared immediately after actuation, and proliferation
was measured after 48-h incubation in a humidified incubator. There
was no significant difference between actuated and non-actuated
cells (P=0.11 for the viability assay, P=0.43 for the proliferation
assay).
Cell biochemistry was evaluated qualitatively by analyzing lysates
with MALDI mass spectrometry. FIGS. 6(a) and (b) show spectra of
lysates of actuated cells and non-actuated cells, respectively.
From previous studies of protein content in Jurkat T-cells,.sup.22
we tentatively assigned several peaks, including heat shock protein
(HSP10) 302, macrophage migration inhibitory factor 304, epidermal
fatty-acid binding protein (E-FABP) 306, and peptidyl-prolyl
cis-trans isomerase A 308. As shown, there are no major qualitative
differences between the two spectra, which suggests that actuation
by DMF does not cause catastrophic effects on cell biochemistry. We
note that MALDI-MS is not a quantitative analysis technique (i.e.,
peak heights can vary considerably within multiple spectra of a
single sample) The gene expression of T-cells and other cell types
using quantitative PCR or gene microarray would be more appropriate
quantitative techniques.
Cell Phenotype Assays by DMF.
To illustrate that DMF is compatible with phenotypic assays, a
dose-response toxicology screen was performed using Jurkat T-cells,
shown in FIGS. 7 and 8. Cells were exposed to varying
concentrations of the surfactant, Tween 20 (0.002% to 0.5% (v/v))
(FIG. 7) and then stained with viability dyes (FIG. 8). The
complete assay, from droplet dispensing to the final incubation
with dyes was performed on-chip. 150 nL droplets (.about.1 mm in
diameter) were dispensed via DMF, and after merging and incubation,
resulted in a final .about.450 nL droplet (.about.1.8 mm diameter,
150 .mu.m height). An equivalent assay was implemented in a
384-well plate with the same number of cells (.about.525 cells/well
or droplet) but different sample volume. In the well-plate assays,
5 .mu.L aliquots of each reagent were pipetted into conical wells
(3.3 mm top-, 2 mm bottom diameter) resulting in a final volume of
15 .mu.L (.about.5 mm height) which is in the recommended range for
384-well plates. Hence, well plates required .about.30-fold greater
reagent use than DMF, leading to a much lower cell concentration in
the wells. As described below, this had significant effects on
assay sensitivity.
A fluorescence microplate reader was used to generate dose response
curves for DMF and well plate assays using identical settings (FIG.
9, error bars are 1 standard deviation). As shown in FIG. 9, the
DMF assays (a) had much lower background signals than the
well-plate assay (b), resulting in a much larger signal-to-noise
ratio than the well-based assays (b). As a consequence, the lowest
detectable number of live cells in droplets was .about.10 (a),
compared to .about.200 cells in wells (b). The latter value matches
the general limits of detection listed by the manufacturer for such
assays. One consequence of this difference was the determination of
different 100%--lethal concentrations of Tween 20: .about.0.5%
(v/v) from the DMF assay and .about.0.03% (v/v) from the well plate
assay. The true 100%-lethal concentration was determined
empirically by staining cells exposed to varying concentrations of
Tween-20 and counting them using a hemacytometer. At the
concentrations evaluated here, the fluorescence microplate reader
results generated by the digital microfluidic method (a) were found
to be a much better approximation of the empirical value than the
conventional method (b). Thus, in this assay, the conventional
method over-estimates the toxicity of Tween 20 by more than
15-fold; this is important, as cytotoxicity is widely used by
regulatory agencies in initial screens for determining acceptable
exposure limits, and by the pharmaceutical industry in early drug
discovery.
Another cause of the improved sensitivity in droplet-based assays
is the high cell concentration in .about.nL droplets. The same
number of cells in .mu.L aliquots results in a much lower
concentration and therefore, lower signal-to-noise ratio. In this
experiment, 525 cells yielded 1.2.times.10.sup.6 cells/mL in
droplets, but only 3.5.times.10.sup.4 cells/mL in wells. In
addition, the cross-sectional density of cells in droplets was
higher because of the slightly smaller droplet diameter (.about.1.8
mm) relative to that of the conical wells (2 mm bottom, 3.3 mm
top). If it is assumed that all cells settled to the bottom of each
well or droplet, then the same number of cells was distributed over
an area that was .about.20% smaller in droplets relative to wells,
resulting in a higher signal. It is possible that all cells
sedimented in droplets (150 .mu.m height), while not all cells
sedimented in wells (.about.5 mm height). If this were the case, it
would obviously contribute to the observed differences in detection
limits.
It should be noted that while the assay described above involved
dispensing, translating, merging and mixing of droplets, other
embodiments of cell assays and cell culture in DMF devices can
include droplet splitting. Droplet splitting is implemented to
reduce a droplet size, number of cells in a droplet, etc.
Some cell assays target molecules that cells secrete into their
microenvironment, such as growth factors, signaling molecules, and
metabolic products. Since DMF droplets of cell suspension are
precise, confined volumes where all cell products are preserved,
they are ideal microenvironment for extracellular biochemistry
assays. In these assays, signal is detected from a suspension
medium rather than cells. Suspension medium can be analyzed by
immunoassays or other means. Droplets of cell suspension can
alternatively be removed from a DMF device and analyzed
externally.
The results presented above demonstrate assaying population of
cells of one kind; nevertheless, it is also possible to assay
droplets containing multiple kinds of cells (e.g., different cell
types, or different phenotypes of the same cell type). Droplets
with multiple kinds of cells can be generated by either dispensing
them from reservoirs containing the same mixed population of cells,
or by combining droplets containing one or several kinds of cells.
Combining droplets, merging and mixing, results in larger droplets
which can be split in droplets of desired size.
Concentration of cells in a droplet can be controlled by the
concentration of cells in a source (a device reservoir or an
external reservoir) or by combining droplets of suspended cells
with droplets of cell suspension medium. In this way, concentration
of cells is reduced by the ratio of the combined volumes. Combined
droplet can be split in smaller droplets which can be further
merged with cell suspension medium for additional cell
concentration reduction. By repeating the procedure above, droplets
with single cells can be generated and used in single-cell
assays.
The results described above demonstrate that DMF can be used to
implement cell-based assays with very high performance. With
reduced reagent and cell consumption, and automated liquid
manipulation, DMF devices outperformed standard well plate assays,
and resulted in significant improvements in assay sensitivity. The
above results clearly demonstrate the efficacy of c DMF cell-based
assays for phenotypic screening.
Cells in Suspension Culture
Cell culture entails growing cells in a growth medium under
controlled temperature and atmosphere conditions. For example,
mammalian cells are grown in humidified atmosphere at 37.degree. C.
and 5% CO.sub.2, in cell culture incubators. Growth medium supplies
nutrients and growth factors to cells; its ingredients are cell
type dependant. In standard cell culture, cells grow suspended in
milliliter volumes in cell culture flasks; they are
split/subcultured every 2-3 days and resuspended in a fresh growth
medium.
In one embodiment of this invention we demonstrate: (1) growing
cells in nanoliter-microliter droplets in DMF devices (in a cell
culture incubator), (2) changing media daily, and 3) splitting
cells every 2-3 days. Media change involves adding one or more
droplets of fresh media to a droplet of incubated cells and thereby
partially replenishing growth media. Cells are further incubated in
the combined droplet or in smaller droplets generated by splitting
the combined droplet. Cell subculture or splitting is achieved
similarly to media change by combining (merging and mixing) a
droplet of incubated cells and a droplet of fresh media, splitting
the combined droplet, and repeating this procedure using the split
droplet(s) until a desired cell concentration is reached. Final
droplets are then incubated, while other droplets of suspended
cells generated in the subculturing process are discarded.
Multiplexed Cell Culture/Cells Assays
In a multiplexed assay 100 (shown in FIG. 10), multiple droplets
106 containing one kind or multiple kinds of cells are exposed to
droplets 108 containing one or multiple reagents 104 and are
assayed similarly to the assays described above. Cells in a
suspension and cell-assay reagents can be deposited in the device
either by dispensing them from device reservoirs 102 (cells) and
104 (reagents) or by dispensing them using external means (e.g.,
pipette, robotic dispenser, etc.), not shown herein. A multiplex
device, an example of which is shown in FIG. 10, can also be used
for multiplex cell culture, where cells can be grown and maintained
in multiple droplets.
There are several ways of configuring the reservoirs. In one
configuration of the method and system the reservoirs may be
external to digital microfluidic device and include for example
arrays of pipettes, robotic dispensers, microprinters and
microstamps. Alternatively, the reservoirs could be integrated as
part of the digital microfluidic device, which are in flow
communication with the hydrophobic/dielectric surface above the
array of actuating electrodes. The reservoirs can be containers
integrated as part of the digital microfluidic device.
Alternatively they may include actuating electrodes from said array
of actuating electrodes modified to act as the liquid reservoirs as
shown in FIG. 1 where reservoir electrodes 32, 34, and 36 store
droplets 42, 44, 46 containing cells, reagent, and dye,
respectively.
The reservoirs could be part of a cartridge assembled with the
digital microfluidic device which is in flow communication with the
hydrophobic/dielectric surface above the array of actuating
electrodes.
The droplets are then translated to pre-selected sites on the top
surface of the substrate 114 on which the array of actuating
electrodes 116 is located. Assays in multiple droplets are
performed simultaneously or sequentially in a certain order defined
by the cell assay protocol. For example, a computer controller
interfaced to the device reservoirs and associated dispensing
devices is programmed to dispense droplets of the suspension of
cells and droplets of one or more cell assay reagents onto the top
surface of the dielectric layer covering the electrode array 116
and surface of the substrate 114, and translating them over said
array of actuating electrodes for mixing the droplets in selected
positions on the array of actuating electrodes to form one or more
secondary droplets in a selected order defined by a selected cell
assay protocol for which said computer controller is
programmed.
Signals from secondary droplets are detected using multiplexed
detection instruments such as optical sensors, optical detectors
comprising a light source and a photodetector, optical detectors
that measure absorbance, fluorescence, epifluorescence,
chemiluminescence, UV light detector, radiometric detector,
scanning, imaging, and confocal microscopy detectors, CCD cameras,
and microplate readers. The detection step is to detect or identify
any reaction products formed by the cell assay, or to identify,
monitor and count the cells if a cell culture is being performed to
mention just a few.
The detection step may be conducted by first translating the
secondary droplet(s) to one or more selected positions on the
substrate surface for analysis or the secondary droplet(s) may be
removed from the device and analyzed externally.
All waste liquid droplets generated during the assay are translated
to the waste container 120. Reservoirs 122 may contain wash
solutions for cleaning the surface of the device between
assays.
Experimental
The use of the digital microfluidics for conducting droplet-based
cell assays using digital microfluidics will now be illustrated
with the following non-limiting examples/studies. More
particularly, herebelow, it is shown experimentally that the
effects of actuation by digital microfluidics on cell vitality are
minimal, and in addition, it is shown that a cytotoxicity assay
implemented by DMF has much better sensitivity than macroscale
methods, which suggests applications in regulatory policy and in
drug discovery. It is also demonstrate compatibility of DMF cell
assays with fluorescence microplate reader detection. This
technique has great potential as a simple yet versatile analytical
tool for implementing cell-based assays on the microscale.
Reagents and Materials.
Unless otherwise indicated, reagents used outside of the clean room
were purchased from Sigma-Aldrich (Oakville, ON), and cells and
cell culture reagents were from American Type Culture Collection
(ATCC, Manassas, Va.). Fluorescent dyes were from
Invitrogen-Molecular Probes (Eugene, Oreg.), Parylene-C dimer was
from Specialty Coating Systems (Indianapolis, Ind.), and Teflon-AF
was purchased from DuPont (Wilmington, Del.). Clean room reagents
and supplies included Shipley S1811 photoresist and MF-321
developer from Rohm and Haas (Marlborough, Mass.), solid chromium
and gold from Kurt J. Lesker Canada (Toronto, ON), standard gold
etchant from Sigma-Aldrich, CR-4 chromium etchant from Cyantek
(Fremont, Calif.), AZ-300T photoresist striper from AZ Electronic
Materials (Somerville, N.J.), and hexamethyldisilazane (HMDS) from
Shin-Etsu MicroSi (Phoenix, Ariz.). Concentrated sulfuric acid and
hydrogen peroxide (30%) were from Fisher Scientific Canada (Ottawa,
ON), and piranha solution was prepared as a 3:1 (v/v) mixture of
sulfuric acid and hydrogen peroxide.
Cell Culture.
Jurkat T-cells (human leukemia lymphocytes) were maintained in a
humidified atmosphere (5% CO.sub.2, 37.degree. C.) in RPMI 1640
medium supplemented with 10% fetal bovine serum (Invitrogen Canada,
Burlington, ON), penicillin (100 IU/mL), and streptomycin (100
.mu.g/mL). Cells were subcultured every 3-4 days at
.about.1.times.10.sup.6 cells/mL. A working buffer of 0.2% (wt/v)
pluronic F68 (Sigma-Aldrich) in Dulbecco's phosphate buffered
saline (PBS) (Invitrogen Canada) was used for most cell-based
assays. Prior to experiments, cells were washed three times in PBS,
suspended in 0.2% F68 (wt/v) in PBS at 3.5.times.10.sup.6 cells/mL,
and then incubated at room temperature (1 h). Cell numbers and
viability were quantified using a hemocytometer and trypan blue
exclusion (Invitrogen Canada) immediately prior to all experiments.
Prior to cell viability/proliferation assays and analysis by mass
spectrometry, cells were incubated for 1 h in 3% (wt/v) F68 in PBS
at 7.2.times.10.sup.6 cells/mL and at 6.times.10.sup.7 cells/mL,
respectively.
Device Fabrication and Use.
Digital microfluidic devices were fabricated using conventional
microfabrication methods. 100 nm thick gold electrodes were
patterned on the bottom plate of a device (glass wafer) and coated
with 2 .mu.m of Parylene-C and 50 nm of Teflon-AF. Unpatterned
indium-tin oxide (ITO) coated glass substrates were coated with 50
nm of Teflon-AF. Devices were assembled with an unpatterned
ITO-glass top plate and a patterned bottom plate and separated by a
.about.150 .mu.m thick spacer. Driving potentials (100-140
V.sub.RMS) were generated by amplifying the output of a function
generator operating at 15 kHz. Droplets were sandwiched between the
two plates and actuated by applying driving potentials between the
top reference electrode 22 and sequential electrodes 14 on the
bottom plate (FIG. 2(a)) via the exposed contact pads. Droplet
actuation was monitored and recorded by a CCD camera mated to a
stereomicroscope with fluorescence imaging capability. Most devices
used here had a geometry identical to that shown in FIG. 2(a) (or
FIG. 1), with 1 mm.times.1 mm actuation electrodes (suitable for
manipulating 150 nL droplets), and inter-electrode gaps of 5-40
.mu.m. The reservoirs were 2 mm.times.2 mm electrodes. Some devices
had 7 mm.times.7 mm actuation electrodes which were used to
manipulate much larger droplets (11 .mu.L).
Electrical Field Modeling.
Electrical fields in digital microfluidic devices were modeled with
COMSOL Multiphysics 3.3a (COMSOL, Burlington, Mass.) using the
conductive media direct current module and the electrostatics
module, shown in FIG. 4. The two-dimensional geometry of the model
was nearly identical to the device illustrated in FIG. 2, including
three patterned electrodes (1 mm length) on the bottom plate, a
layer of Parylene-C (2 .mu.m thick), a layer of PBS and air (150
.mu.m thick), and a continuous electrode on the top plate. The
hydrophobic Teflon AF layer 18 was omitted from the model because
of its porosity and insignificant thickness. Dielectric constants,
.epsilon., and conductivities, .sigma., used in the model included
.epsilon..sub.parylene=2.65, .epsilon..sub.pbs=70,
.epsilon..sub.air=1, .sigma..sub.parylene=0 S/m, .sigma..sub.air=0
S/m, and .sigma..sub.pbs=4.7 S/m (measured using a conductivity
meter). With a 100 V potential applied between the bottom-right
electrode and the top electrode (ground), a mesh with 233,831
triangular elements was used to simulate electrical field, using
the linear solver UMFPACK.
Vitality Assays.
The effects of the electric field driven droplet actuation on cell
vitality were evaluated by three assays, measuring cell viability
(FIG. 5 day 0), proliferation (FIG. 5 day 2), and biochemistry
(FIG. 6). In these vitality assays, large droplets (>1 .mu.L)
were used because the more conventional sub-microliter droplets
(used in the cell phenotype assays) were difficult to handle
off-chip and did not contain enough cells for analysis. In the cell
viability and proliferation assays, ten 11 .mu.L droplets of cells
suspended in PBS/F68 (each containing .about.79,200 cells) were
actuated on devices with 7.times.7 mm electrodes. Each droplet was
moved across 10 electrodes (approximately 15 s of actuation per
droplet) and was then removed from the device and suspended in 300
.mu.L of cell medium at 2.5.times.10.sup.5 cells/mL. For viability
assays, immediately after suspension in media, live and dead cells
were counted on a hemacytometer with trypan blue exclusion. For
proliferation assays, live and dead cells were counted after 48 h
of incubation off-chip (humidified incubator, 5% CO.sub.2,
37.degree. C.). A second group of ten 11 .mu.L droplets of the
original cell solution (in PBS/F68) were treated identically, but
were not actuated, and served as a control. The data was analyzed
with two-tailed t-test assuming unequal variances.
In the cell biochemistry assay, four 11 .mu.L droplets of cell
suspension (.about.6.6.times.10.sup.5 cells/droplet) were actuated
over ten electrodes as above, and were then pooled and suspended in
lysing medium at 3.times.10.sup.7 cells/mL. Lysing medium was PBS
with 3% (wt/v) F68, 1% Triton X-100, and 1 mM phenylmethylsulphonyl
fluoride (PMSF). After incubation on ice (30 min), the lysate was
centrifuged (12,000 rpm, 5 min) and the supernatant was collected
and stored in a -85.degree. C. freezer. Immediately prior to
analysis, the supernatant (100 .mu.L) was thawed and desalted using
a microspin G-25 column (Amersham BioSciences, Piscataway, N.J.) at
2800 rpm for 2 min. Proteins were eluted in distilled water with
0.05% (v/v) Kathon (1.5 .mu.L), and the eluent was spotted onto a
MALDI (matrix assisted laser desorption/ionization) target plate. A
1.5 .mu.L aliquot of MALDI matrix solution (10 mg/mL sinapinic acid
in 80% (v/v) acetonitrile/water) was added and the combined droplet
was allowed to dry. Non-actuated droplets of the original cell
suspension were lysed and processed identically, and served as a
control.
Samples were analyzed using a MALDI-TOF Micro MX mass spectrometer
(Waters, Milford, Mass.) in linear positive mode for the mass range
of 4,000 to 25,000 m/z. One hundred shots were collected per
spectrum, with laser power tuned to optimize the signal over noise
ratio. Data were then processed by normalization to the largest
analyte peak, baseline subtraction, smoothed with a 15-point
running average.
Cell Phenotype Assays.
For phenotypic assays, cells were exposed to the surfactant, Tween
20 (lethal to mammalian cells at high concentrations), diluted in
working buffer in a range of concentrations (0.002% to 0.5%
(wt/vol)). Each Tween 20 concentration was evaluated in 4-6
replicates. In each experiment, a 150 nL droplet containing
.about.525 cells was dispensed and merged with a 150 nL droplet
containing Tween 20. The merged droplets were then actively mixed
by moving them on four neighboring electrodes in a circle. After 20
min of incubation in a humidified environment (a closed petri dish
half-filled with water), the combined droplet containing cells and
Tween 20 was merged and mixed with a 150-nL probe droplet
containing viability dye(s), and then incubated for a second time
in a humidified environment (20 min). In all experiments, the probe
droplet contained calcein AM (1 .mu.M in the working buffer), and
in some experiments, the droplet also contained ethidium
homodimer-1 (2 .mu.M in the working buffer).
For quantitative experiments, a digital microfluidic device was
positioned on the top of a well plate and inserted into a
fluorescence microplate reader (Pherastar, BMG Labtech, Durham,
N.C.) equipped with a module for 480 nm excitation and 520 nm
emission. Each droplet was evaluated using a multipoint scanning
program, in which the average fluorescence was recorded from each
of 9 excitation flashes illuminated onto a 1-mm square 3.times.3
array with 0.5 mm resolution. The array was located in the centre
of each droplet, and the focal height was set for each analysis at
the highest-signal intensity, with gain=376. This multipoint
program, designed by BMG Labtech for standard assays in well
plates, was found empirically to have lower variance between runs
than comparable single point analyses. Samples containing only
Tween 20, pluronic F68, and calcein AM in PBS were evaluated to
determine the background signal. Each analysis was repeated 4-6
times to determine standard deviations. All data were normalized to
the average fluorescence intensity of cell samples exposed to
control droplets (containing no Tween-20), and were plotted as a
function of Tween-20 concentration.
For comparison, each assay implemented by digital microfluidics was
duplicated in standard 384-well plates by pipetting reagents,
cells, and dyes. In these experiments, all parameters were
identical to those described above, except that the .about.525
cells, reagents, and dyes were suspended in a final volume of 15
.mu.L.
Culturing and Assaying Adherent Cells
The majority of mammalian cells are adherent, i.e. anchorage
dependent. In a further embodiment of the present invention, we
demonstrate that DMF can also be used to culture and assay adherent
cells. In in vitro conditions, adherent cells grow in layers
attached to a substrate that is typically hydrophilic and
negatively charged, such as tissue culture treated polystyrene.
Cells are maintained/grown in cell culture (growth) media in
incubators with humidified atmosphere at 37.degree. C. and with 5%
CO.sub.2.
As shown in FIGS. 11a, 11b, 11c, and 11d, the surface of a DMF
device 200 (specifically the hydrophobic surface 18 that covers the
dielectric material 16 on the lower electrode 14 (see FIG. 2(a)) is
modified in specific areas, cell culture sites (CCS) 202, to
facilitate cell adhesion and proliferation (cell growth and
division). The surface modification procedure reported here makes
use of standard techniques, such as depositing (microprinting,
micorstamping) a bio-substrate (typically extracellular matrix
proteins 206), rendering a hydrophilic and charged surface via
microfabrication, or any other surface modification procedure that
can also be cell specific.
In addition to using standard techniques, a bio-substrate can be
formed by dispensing a droplet of a bio-substrate solution in a DMF
device and translating it to the cell culture site 202, where after
incubation and drying, it forms a bio-substrate layer for cell
attachment. In this case, a device has an extra reservoir holding
the bio-substrate solution. After the cell culture site 202 is
formed, cells are seeded by generating a droplet 214 of growth
media with suspended cells 212 on the cell culture site CCS 202
(FIG. 11c). Cells are allowed to adhere to the surface forming a
cell monolayer 204 (FIG. 11d).
There are two ways of generating a droplet 214 on the cell culture
sites 202: (1) by actively dispensing a droplet from a device
reservoir or via external means (e.g. pipetting) and translating
the droplet to the cell culture sites 202 (FIG. 11(a)), and (2) by
actuating a droplet 216 (source droplet) larger than the cell
culture sites 202 over the cell culture sites 202 and thereby
passively dispensing the desired droplet on the hydrophilic cell
culture sites 202 (FIG. 11(b)). Passive dispensing will be
described in more details in the following section.
Passive Dipensing, Passive Washing, Passive Media/Reagent
Exchange
Referring to FIG. 12, when a source droplet 210 is actuated in a
DMF device over a patterned hydrophilic area 201 smaller than the
base area of the source droplet 210, it leaves behind a smaller
droplet 230 on the hydrophilic area 201 and the rest of source
droplet 210 is translated away from droplet 230. This method of
generating droplets is termed passive dispensing. Methods for
producing the hydrophilic areas 201 include but are not limited to
microfabrication techniques (e.g. exposing hydrophilic layers of a
device, such as glass or electrodes, in specific areas),
hydrophobic surface plasma treatment, or deposition of a thin,
patterned, hydrophilic layer onto a device surface. Hydrophilic
areas can be formed on either the top plate, the bottom plate, or
both the top and bottom plate of a two plate device. In the
applications disclosed herein of adherent cell culture and
assaying, hydrophilic areas 201 are used as the cell culturing
sites (indicated by reference numeral 202 in FIG. 11) which
preferably patterned by depositing bio-substrates, made from cell
specific constituents, such as, but not limited to, extracellular
matrix (ECM) proteins. ECMs are more favorable substrate for cell
attachment than bare glass, electrodes, or a dielectric layer.
Examples of extracellular matrix proteins include, but are not
limited to fibronectin, laminin, collagen, elastin. The cell
specific constituents may also comprise synthetic molecules
comprised of one of poly-L-lysine, poly-D-lysine and any
combination thereof for example.
Typically, there are no electrodes underneath hydrophilic areas, as
these areas (inherently hydrophilic) do not need to be electrically
addressed to attract droplets; however, they have to be at least in
the vicinity of electrodes. It will be appreciated that the
hydrophilic arrays can also be formed on the top surface of the
layer coating electrodes right above electrodes themselves. In most
cell-based applications, it is desirable to have transparent
attachment substrate to enable facile cell visualization.
Referring to FIG. 13, the size and position of a hydrophilic area
can vary relative to size and position of electrodes 14 for source
droplets actuation. Two relative sizes of hydrophilic areas--1/4
and 1/9 of the electrode size were studied, and several positions
relative to electrodes 14 and to a source droplet path. It should
be noted that size and position of hydrophilic areas 201 is not
limited by the examples in FIG. 13, and that the shape of
hydrophilic areas 201 and actuating electrodes 14 is not limited to
the square shape.
Referring to FIG. 14, when a hydrophilic area 201 is already
occupied by a droplet 230, a source droplet 210 will remove the
smaller droplet 230 and replace it with a new droplet 232 of the
source solution while removing droplet 230 in droplet 210'. This
process is termed passive washing or passive exchange of liquid
solutions on hydrophilic areas 201 (e.g., on CCSs) in a DMF device.
We report passive exchange efficiency of .gtoreq.95% with a single
source droplet, or .gtoreq.99% with two or more consecutive source
droplets. FIG. 15 shows efficency of 0.5 nM fluorescein passive
exchange with phosphate buffered saline. These results were
obtained with fibronectin hydrophilic areas 201, .about. 1/9 of the
electrode size, having two different positions relative to
actuating electrodes 14.
Culturing and Passaging Adherent Cells
For adherent cell culture, a DMF device with seeded cells is placed
in a cell culture incubator and a droplet of culture media on top
of the cell layer 204 is regularly replenished with fresh media via
DMF passive exchange every 24 h. We report culturing cells on cell
culture sites 202 for 72 h; growth characteristics and morphology
of the cells are comparable to cells grown in standard tissue
culture flasks (FIG. 16). No detachment of cells was observed
during media droplet actuation over the cell culture sites 202.
Cells are subcultured at regular intervals using standard
subculturing protocols adapted to DMF system: (1) washing cells as
shown in FIG. 17(b) in which washing droplet 213 has been dispensed
and translated over cell culture site 202, (2) harvesting cells by
dispensing and translating a droplet 215 containing a dissociation
agent (e.g. trypsin, collagenase) over cell culture site 202 as
shown in FIG. 17(c) and incubating to detach the adhered cells and
resuspend them as shown in FIG. 17(d), (3) a droplet 240 containing
a blocking agent (typically serum in cell culture media) for
blocking the dissociation agent is dispensed and translated over
cell culture site 202, while removing the detached cells away from
the cell culture sites 202 as shown in FIG. 17(e), (4) splitting
the resulting cell suspension as necessary and resuspending in
fresh media in droplet 242 and (5) seeding resuspended cells on a
new cell culture site 202 as shown in FIG. 17(f). Blocked
dissociation agent and cell suspension are diluted in a big source
droplet 240 of a blocking agent (cell culture media with serum) by
the ratio of the volumes of the two droplets, cell culture site 202
droplet and the source droplet. In step (4), the resulting cell
suspension can be split in smaller droplets and resuspended in
droplets of fresh media for further reduction of cell
concentration. When a desired cell concentration is achieved, new
generation of cells is seeded on new cell culture sites 202 by
either translating actively dispensed droplets of the cell
suspension to new cell culture sites, or by passively dispensing
droplets with cells on cell culture sites 202 from droplet 242
(FIG. 17f). The inventors have demonstrated subculturing several
generations of mammalian cells in the same DMF device following the
procedure outlined above.
Assaying Adherent Cells
Adherent cell assays in DMF devices are executed in droplets on
cell culture sites 202 where adherent cells are seeded. Devices
with seeded cells are placed in incubators for few hours or
overnight to allow cell attachment and adjustment to a new DMF
device environment (FIG. 18a). When adherent cell deposits 204 are
ready for assaying, droplets of reagents and washing solutions are
deposited on cell culture sites 202 either by translating a droplet
actively dispensed from a device reservoir or externally, or by
passive dispensing/exchange from source droplets 250 (FIG. 18b).
Source droplets 250 are either dispensed via DMF from reservoirs or
externally deposited on a device. Washing solutions and reagents
are incubated with cells following cell assay protocols (FIG. 18c).
Upon assay completion, cell response to a stimulus (e.g. a lead
drug compound) can be detected and measured by apparatus 260 which
may be any standard means (e.g. fluorescence microscopy, microplate
reader to give a few examples) (FIG. 18d).
In assays targeting extracellular biochemistry (growth factors,
signaling molecules, metabolic products, etc.), cell response to
stimulus is detected in medium where cells are grown and stimulated
with reagents, rather than in cells. Medium can be analyzed by
immunoassays or other means. Droplets of cell suspension can
alternatively be removed from the cell culture sites 202 (e.g. with
a bigger source droplet) and its signal can be detected on another
spot or its contents can be analyzed externally.
Multiplexed Adherent Cell Culture/Cell Assays
Referring to FIG. 19, multiple cell culturing sites 202 in a DMF
device 300 which is similar to device 100 in FIG. 10 but device 300
includes a plurality of cell culture sites 202. Device 300 may be
used in multiplexed assays where cells of one kind or multiple
kinds are assayed with one or multiple reagents simultaneously in
which cell culturing may be involved as well. In addition, a single
cell culture site 202 can be seeded with multiple cell lines (cell
co-culture). Assay reagents and/or culture media can be delivered
to cell culture sites 202 via passive dispensing/exchange or in
actively dispensed droplets.
In a multiplexed assay, a single source droplet can deliver
reagents to multiple cell culture sites 202 (serial passive
dispensing/exchange), or to only one cell culture site 202
(parallel passive dispensing/exchange). Signals from assayed cells
or suspension media is detected using multiplexed detection
instruments such as microplate readers.
Experimental
The following non-limiting examples demonstrates the efficacy of
the present invention for conducting adherent cell assays and
culture.
Device Design and Fabrication.
Digital microfluidic devices were fabricated using conventional
microfabrication methods. 100 nm thick gold electrodes were
patterned on the bottom plate of a device (glass wafer) and coated
with 2 .mu.m of Parylene-C and 50 nm of Teflon-AF. Unpatterned
indium-tin oxide (ITO) coated glass substrates were coated with 50
nm of Teflon-AF. Devices were assembled with an unpatterned
ITO-glass top plate and a patterned bottom plate and separated by a
.about.150 .mu.m thick spacer. Driving potentials (100-140
V.sub.RMS) were generated by amplifying the output of a function
generator operating at 15 kHz. Droplets were sandwiched between the
two plates and actuated by applying driving potentials between the
top reference electrode 22 and sequential electrodes 14 on the
bottom plate (FIG. 2(a)) via the exposed contact pads. Most devices
had a basic geometry identical to that shown in FIG. 11 with the
addition of reservoirs. Source droplets (.about.800 nL) were
actuated on 2.5 mm.times.2.5 mm actuation electrodes, and smaller
droplets were actuated on 0.8 mm.times.0.8 mm actuation electrodes.
Cell culture site (CCS) areas were patterned either as transparent,
non-conductive fields in 2.5 mm.times.2.5 mm electrodes or as
smaller (0.8 mm.times.0.8 mm) electrodes within the area of larger
2.5 mm.times.2.5 mm electrodes. Devices were sterilized in 70%
ethanol prior to use.
Cell Culture
NIH-3T3 cells (mouse fibroblasts) were maintained in a humidified
atmosphere (5% CO.sub.2, 37.degree. C.) in DMEM supplemented with
10% fetal bovine serum, penicillin (100 IU mL.sup.-1), and
streptomycin (100 .mu.g mL.sup.-1). Cells were subcultured every
2-3 days at 5.times.10.sup.3 cells cm..sup.-2 Prior to each DMF
experiment, cells were suspended in DMEM with the addition of 0.05%
(wt/v) pluronic F68 (Sigma-Aldrich) at .about.7.times.10.sup.5
cells mL..sup.-1 Cell number and viability were quantified using a
hemocytometer and trypan blue exclusion (Invitrogen Canada)
immediately prior to all experiments.
DMF Cell Seeding
CCSs were formed by depositing 500 nL droplets of fibronectin (100
.mu.g mL.sup.-1 in ddH.sub.2O) on designated areas in DMF devices.
Fibronectin solution was air-dried resulting in .about.1 mm.sup.2
bio-substrates with .about.5 .mu.g/cm.sup.2 of fibronectin. Cell
suspension was delivered to CCSs by either passive dispensing from
a source droplet or by translating actively dispensed droplets from
a device reservoir to CCSs. CCS droplets were .about.200 nL in
volume and contained .about.140 cells. Cells were allowed to attach
to the substrate and adapt overnight in a cell culture incubator
(5% CO.sub.2, 37.degree. C.).
DMF Cell Culture
NIH-3T3 cells were maintained on CCSs by changing media via passive
dispensing every 24 hours. Complete DMEM containing 0.05% (wt/v)
pluronic F68 was dispensed in .about.800 nL droplets and translated
over CCSs while replenishing CCS droplet of media. Complete media
exchange was accomplished with two consecutive source droplets and
cells were returned to the incubator. No cell detachment was
observed during passive media exchange.
DMF Cell Subculture
Upon reaching confluency on CCSs, cells were subcultured following
standard subculturing protocols adapted to the DMF format. All
reagents and media containing 0.05% (wt/v) pluronic F68 were
delivered to cells using passive dispensing/exchange from two
consecutive source droplets. Cells were first washed with PBS
without Ca.sup.2+/Mg.sup.2+ and then supplied and incubated with
GIBCO Trypsin-EDTA dissociation agent (0.25% Trypsin, 1 mM EDTA
4Na) for 5-10 min at 37.degree. C. DMEM source droplet was then
translated to the CCS to block the dissociation agent with the
serum present in media, whereby harvested cells were resuspended in
DMEM droplet at the 1:4 ratio. DMEM droplet with suspended cells
was actuated away from the CCS and used either as a source droplet
or a reservoir droplet to seed the new generation of cells on a new
CCS in the same device. Seeded cells were placed in a cell culture
incubator overnight followed by media change. Cells were grown on
the new CCS for 2 days and further subcultured on the same
device.
DMF Cell Viability Assay
Cells cultured on CCSs were assayed on a device for viability.
Source droplets of 0.05% (wt/v) pluronic F68 (Sigma-Aldrich) in
phosphate buffered saline containing viability dyes, calcein AM (1
.mu.) and ethidium homodimer-1 (2 .mu.M) (Invitrogen Canada), were
dispensed in a device and translated over the CCS. With two
consecutive source droplets, growth media was removed from the CCS
and replaced with viability dyes. Cells were incubated with dyes at
room temperature and visualized using stereomicroscope. Viability
of cells was higher than 95% and there was no significant
difference in morphology between cells grown on CCSs and cells
grown in cell culture flasks.
It will be understood that when doing cell culturing or cell
assaying, the suspension of cells may contain a combination of
cells, a suspension medium, and a non-ionic surfactant. The
suspension medium may be selected to facilitate cell-containing
droplet actuation by preventing non-specific adsorption of cells
and proteins to device surfaces. The suspension of cells may be a
combination of cells and a suspension medium comprised of block
copolymers formed from poly(propylene oxide) and poly(ethylene
oxide), pluronic F68, pluronic F127, hydrophilic polymers, sodium
bicarbonate, phosphate buffered saline (PBS), HEPES, and other
biological buffers, and any combination thereof, which may be
combined or mixed with cell culture medium which in turn may
include balanced salt solutions, nutrient mixtures, basal media,
complex media, serum free media, insect cell media, virus
production media, serum, fetal bovine serum, serum replacements,
antibiotics, antimycotics, and any combination thereof.
In an embodiment the suspension of cells may be a combination of
cells, phosphate buffered saline, and pluronic F68. The droplets
including a cell assay reagent may include chemicals, biochemicals,
drugs, drug lead compounds, toxins, surfactants, transfection
reagents, supplements, cell culture media, anti-clumping agents,
streptavidin, biotin, antibody production enhancers, antibodies,
antibody ligands, nucleic acids, nucleic acid binding molecules,
enzymes, proteins, viruses, cell process agonists or antagonists,
labeling agents, fluorescent dyes, fluorogenic dyes, viability
dyes, calcein AM, quantum dots, nano particles, Tween 20, and
ethidium homodimer-1, block copolymers formed from poly(propylene
oxide) and poly(ethylene oxide), pluronic F68, pluronic F127,
hydrophilic polymers, sodium bicarbonate, phosphate buffered saline
(PBS), HEPES, and other biological buffers, and any combination
thereof, which may be combined or mixed with cell culture medium
which in turn may include balanced salt solutions, nutrient
mixtures, basal media, complex media, serum free media, insect cell
media, virus production media, serum, fetal bovine serum, serum
replacements, antibiotics, antimycotics, and any combination
thereof.
The cells in the suspension of cells may include primary/isolated
or transformed/cultured cells selected from the group consisting of
various eukaryotic and prokaryotic cells, including animal cells
(blood cells, human leukemia cells, lymphocytes, beta cells,
oocytes, eggs, primary cells, primary bone marrow cells, stem
cells, neuronal cells, endothelial cells, epithelial cells,
fibroblasts), insect cells, plant cells, bacterial cells,
archebacterial cells.
As used herein the word "incubation" can mean allowing a reaction
to take place over a period of time under specified conditions. For
cell assays involving mixing of cells with one or more cell assay
reagents, the incubation period may be very short or almost
instantaneous upon mixing the droplets wherein the reaction or
response of the cells to the reagent occurs quickly. For cell
culture, "incubation" can mean maintaining the cells growing or
alive under specific conditions and the period of time of the
"incubation" may be arbitrary, after which point the cells may be
subcultured, assayed or subject to further culturing.
The results disclose herein demonstrate the utility of the present
invention for its application of digital microfluidics to
multiplexed, high throughput, phenotypic cell-based assays, an
important tool used in drug discovery and environmental monitoring.
To facilitate high-throughput screening, arrays of DMF cell culture
sites (FIG. 19) can be addressed with compounds from chemical
libraries, and the potential drugs evaluated on the basis of
observed phenotypic changes. The proposed method will enable
high-throughput phenotypic screening with 100-1000.times. lower
reagent consumption than conventional methods; in addition, the
devices are inexpensive (relative to robotic dispensers), have
small laboratory footprint and no moving parts. This method could
transform high-throughput screening, making it attractive to
pharmaceutical companies and accessible for basic and applied
scientists, world-wide.
In addition to cell assaying the inventors disclose herein the
first multigenerational lab-on-a-chip cell culture using DMF
devices. Cells are grown, maintained and subcultured in nanoliter
volumes. DMF devices are inherently easily automated and as such
have a high potential to be used as tool for a completely automated
microscale cell culture system.
As used herein, the terms "comprises", "comprising", "includes" and
"including" are to be construed as being inclusive and open ended,
and not exclusive. Specifically, when used in this specification
including claims, the terms "comprises", "comprising", "includes"
and "including" and variations thereof mean the specified features,
steps or components are included. These terms are not to be
interpreted to exclude the presence of other features, steps or
components.
The foregoing description of the preferred embodiments of the
invention has been presented to illustrate the principles of the
invention and not to limit the invention to the particular
embodiment illustrated. It is intended that the scope of the
invention be defined by all of the embodiments encompassed within
the following claims and their equivalents.
References
(1) Verkman, A. S., "Drug discovery in academia," American Journal
of Physiology-Cell Physiology 2004, 286, C465-C474. (2) El-Ali, J.,
Sorger, P. K., Jensen, K. F., "Cells on chips," Nature 2006, 442,
403-411. (3) Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A.,
Quake, S. R., "Monolithic Microfabricated Valves and Pumps by
Multilayer Soft Lithography," Science 2000, 288, 113-116. (4) Yu,
H. M., Alexander, C. M., Beebe, D. J., "A plate reader-compatible
microchannel array for cell biology assays," Lab on a Chip 2007, 7,
388-391. (5) Le Pesant, J.-P., 1987, U.S. Pat. No. 4,636,785. (6)
Ohkawa, T., 1996, U.S. Pat. No. 5,486,337. (7) Washizu, M.,
Kurosawa, O., 1998, Japan 10267801. (8) Washizu, M., "Electrostatic
Actuation of Liquid Droplets for Microreactor Applications," IEEE
Transactions on Industry Applications 1998, 34, 732-737. (9) Lee,
J., Moon, H., Fowler, J., Schoelihammer, T., Kim, C.-J.,
"Electrowetting and electrowetting-on-dielectric for microscale
liquid handling," Sensors & Actuators A 2002, 95, 259-268. (10)
Pollack, M. G., Fair, R. B., Shenderov, A. D.,
"Electrowetting-based actuation of liquid droplets for microfluidic
applications," Applied Physics Letters 2000, 77, 1725-1726. (11)
Shenderov, A. D., 2003, U.S. Pat. No. 6,565,727. (12) Shenderov, A.
D., 2007, U.S. Pat. No. 7,255,780. (13) Elrod, S. A., Peeters, E.
T., Biegelsen, D. K., Dunec, J. L., 2006, U.S. Pat. No. 7,147,763.
(14) Pamula, V. K., Pollack, M. G., Paik, P., H., R., Fair, R.,
2005, U.S. Pat. No. 6,911,132. (15) Chen, T.-H., Su, C.-M., Shih,
H.-C., Yang, C.-T., "Selective Wettability Assisted Nanoliter
Sample Generation via Electrowetting-Based Transportation,"
Proceedings of the Fifth International Conference on Nanochannels,
Microchannels and Minichannels (ICNMM2007), Puebla, Mexico, Jun.
18-20 2007. (16) Pollack, M., G., Pamula, V., K., Srinivasan, V.,
Paik, P., Y., Eckhardt, A., E., Fair, R., B., 2007 WO/2007/120241.
(17) Huh, N., Lee, J.-g., 2007, US 20070148763 (18) Fan, S.-K.,
Huang, P.-W., Wang, T.-T., Peng, Y.-H., "Cross-scale electric
manipulations of cells and droplets by frequency-modulated
dielectrophoresis and electrowetting," Lab on a Chip 2008,
10.1039/b803204a. (19) Smith, C. M., Hebbel, R. P., Tukey, D. P.,
Clawson, C. C., White, J. G., Vercellotti, G. M., "Pluronic F-68
Reduces the Endothelial Adherence and Improves the Rheology of
Liganded Sickle Erythrocytes," Blood 1987, 69, 1631-1636. (20)
Mizrahi, A., "Pluronic Polyols in Human Lymphocyte Cell Line
Cultures," Journal of Clinical Microbiology 1975, 2, 11-13. (21)
"Hyclone Media: CHO Cell Culture Plafform Media,"
http://www.hyclone.com/media/cho.htm, accessed in 2007. (22)
Thiede, B., Siejak, F., Dimmler, C., Jungblut, P. R., Rudel, T., "A
two dimensional electrophoresis database of a human Jurkat T-cell
line," Electrophoresis 2000, 21, 2713-2720.
* * * * *
References